Targeting epidermal growth factor receptor (EGFR) activating mutations, 90% of which present as an exon 19 deletion (Del19) or exon 21 point mutation (L858R), with first generation EGFR tyrosine kinase inhibitors (EGFR-TKIs; e.g., erlotinib and gefitinib) and the T790M resistance mutation with third generation EGFR-TKIs (e.g., AZD9291; TAGRISSOTM or osimertinib) has provided significant clinical benefit in patients with non-small cell lung cancer (NSCLC) harboring these mutations. Unfortunately, resistance to these EGFR-TKIs occurs in the clinic, resulting in disease progression. Hence, understanding the underlying mechanisms and developing effective strategies to overcome EGFR-TKI resistance is highly desirable and urgently needed in the clinic. Death receptor 4 (DR4; also known as TRAIL-R1 or TNFRSF10A) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). It is generally thought that its activation, upon binding to TRAIL, induces apoptosis; this function is recognized as a critical mechanism of immunosurveillance against cancer cells. However, whether DR4 exerts other as yet unknown biological functions is unclear. Our preliminary data demonstrated that EGFR inhibition with several EGFR inhibitors including AZD9291, CO1686, and erlotinib substantially decreased DR4 levels primarily in EGFR-TKI-sensitive EGFR-mutant NSCLC cell lines; this DR4 reduction is tightly associated with ERK suppression and induction of apoptosis in the tested cell lines. Unexpectedly, knockdown of DR4 augments TRAIL-induced apoptosis and also enhances AZD9291- induced apoptosis. This proposal thus will test the overall hypothesis that suppression of DR4 expression is an on-target consequence of EGFR inhibition and has critical impact on both early and long-term therapeutic efficacy of EGFR-targeted therapy. This hypothesis will be tested by accomplishing the following specific aims: (1) to demonstrate the mechanism(s) by which EGFR inhibitors suppress DR4 expression; (2) to understand the biological significance of DR4 suppression in EGFR-targeted cancer therapy; and (3) to elucidate the effect of DR4 downregulation on the efficacy of EGFR-targeted therapy using preclinical NSCLC patient-derived xenografts (PDXs) and samples from NSCLC patients treated with EGFR-TKIs. The objectives of this proposal are to demonstrate the molecular mechanism underlying DR4 reduction caused by EGFR inhibition, to understand the biological significance of DR4 suppression in EGFR-targeted cancer therapy, and to validate DR4 downregulation in clinical specimens from NSCLC patients receiving EGFR-TKIs. We believe that the outcomes of this proposal will be of high translational significance with immediate impact on EGFR-targeted cancer therapy in the clinic. 1